Molecular biology comprises a wide variety of techniques for the analysis of nucleic acid and protein. Many of these techniques and procedures form the basis of clinical diagnostic assays and tests. These techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and the separation and purification of nucleic acids and proteins (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed., Cold spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Most of these techniques involve carrying out numerous operations (e.g., pipetting, centrifugations, electrophoresis) on a large number of samples. They are often complex and time consuming, and generally require a high degree of accuracy. Many a technique is limited in its application by a lack of sensitivity, specificity, or reproducibility. For example, these problems have limited many diagnostic applications of nucleic acid hybridization analysis.
The complete process for carrying out a DNA hybridization analysis for a genetic or infectious disease is very involved. Broadly speaking, the complete process may be divided into a number of steps and substeps, broadly including the steps of obtaining the sample, disrupting the cells within the sample, performing complexity reduction or amplification, performing some sort of assay or hybridization, followed by detection of the presence or absence of a desired event serving to generate a result.
New techniques are being developed for carrying cut multiple sample nucleic acid hybridization analysis on micro-formatted multiplex or matrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). These methods usually attach specific DNA sequences to very small specific areas of a solid support, such as micro-wells of a DNA chip. These hybridization formats are micro-scale versions of the conventional "dot blot" and "sandwich" hybridization systems.
A variety of methods exist for detection and analysis of the hybridization events. Depending on the reporter group (fluorophore, enzyme, radioisotope, etc.) used to label the DNA probe, detection and analysis are carried out fluorometrically, colorimetrically, or by autoradiography. By observing and measuring emitted radiation, such as fluorescent radiation or particle emission, information may be obtained about the hybridization events. Even when detection methods have very high intrinsic sensitivity, detection of hybridization events is difficult because of the background presence of non-specifically bound materials and materials with inherent fluorescent characteristics. A number of other factors also reduce the sensitivity and selectivity of DNA hybridization assays.
In conventional fluorometric detection systems, an excitation energy of one wavelength is delivered to the region of interest and energy of a different wavelength is emitted and detected. Large scale systems, generally those having a region of interest of two millimeters or greater, have been manufactured in which the quality of the overall system is not inherently limited by the size requirements of the optical elements or the ability to place them in optical proximity to the region of interest. However, with small geometries, such as those below 2 millimeters, and especially those on the order of 500 microns or less in size of the region of interest, the conventional approaches to fluorometer design have proved inadequate. Generally, the excitation and emission optical elements must be placed close to the region of interest. Preferably, a focused spot size is relatively small, often requiring sophisticated optical designs. As the size of the feature to be observed decreases, the demands for high accuracy in mechanical alignment increase. Further, because it is usually desirable to maximize the detectable area, the size of the optical components required to achieve these goals in relation to their distance from the region of interest becomes important, and in many cases, compromises the performance obtained.
Various prior art attempts have been made to image multiple sites in immunoassay systems. In Leaback, U.S. Pat. No. 5,096,807, there is a disclosure of an imaging immunoassay detection apparatus system and method purported to be capable of detecting and quantifying multiple light-emitting reactions from small volume samples simultaneously. A plurality of individual chemical reactant samples are each capable of emitting photons when a reaction takes place. These samples are arranged in a spaced relationship with respect to each other, and a detection system is operatively positioned so as to simultaneously detect the presence and x-y location of each photon emitted from any reacting sample. One disclosed carrier is a microtiter plate with multiple samples, e.g., 96, arranged in rows and columns. Various imaging devices arc disclosed, such as an imaging photon detector, microchannel plate intensifiers and charged coupled devices (CCDs). Preferably, the signals representing the discrete areas of reactions have the background noise signal subtracted from them.
Yet other systems for imaging multiple sites in immunoassay systems utilize sequential scanning techniques. Multiple-well screening fluorometer systems move multiple sites relative to a fluorometer. Certain versions of the systems utilize a motorized stage and others arrange the samples on a wheel, which sequentially rotate samples into position for observation by the fluorometer. With these techniques, the samples are presented to the detector in a serial manner.
Another multiple location immunoassay system is disclosed in Elings et al. U.S. Pat. No. 4,537,861 entitled "Apparatus and Method for Homogeneous Immunoassay". A spatial pattern formed by a spatial array of separate regions of antiligand material are disposed on a surface. The presence or absence of a binding reaction between a ligand and the antiligand is then detected. A source of illumination is shined on the combined ligand-antiligand location, and the emitted radiation detected. The contribution to the imager due to free labeled molecules plus background contaminants are suppressed through use of a chopper system in positional correlation to the examined array which generates a reference signal.
Various microscope systems for the detection of fluorescence or chemiluminescence have been known to the art. For example, Dixon et al. U.S. Pat. No. 5,192,980 entitled "Apparatus and Method for Spatially- and Spectrally-Resolved Measurements" discloses a scanning optical microscope or mapping system for spectrally-resolved measurement of light reflected, emitted or scattered from a specimen. A confocal scanning laser microscope system is combined with a grating monochromator located in the detector arm of the system. A spectrally resolved image is generated for a given point of illumination. Spatial resolution is achieved by moving the sample on a movable stage.
Another scanning confocal microscope is disclosed in U.S. Pat. No. 5,296,703 entitled "Scanning Confocal Microscope Using Fluorescence Detection". A scanning confocal microscope is provided for scanning a sample with an incident beam of radiation and detecting the resulting fluorescence radiation to provide data suitable for use in a raster scanned display of the fluorescence. First and second closely spaced scanning mirrors direct an incident beam to a sample and direct the fluorescent radiation towards a fluorescence detection system. Spectral resolution is achieved in the detection system by utilizing a dichroic mirror which serves to separate various wavelengths which are then separately detected by photomultiplier tubes. The system additionally generates a reference beam which impinges on one of the scanning mirrors, the reflected scanning reference beam is directed through a grating and having an alternating sequence of transparent and opaque regions. The transmitted beam is detected and utilized to generate a clock signal representative of the position of the scanning reference beam. The clock signal is used to control analog-to-digital circuits in the fluorescence detection system. In this way, the sampling of the outputs of the photomultiplier tubes generates data representative of linear scans of the sample, despite the use of a scanning mirror that scans in a non-linear, sinusoidal fashion.
Despite the desirability of having an improved examining system, and the need for higher sensitivity in such systems, the systems described previously have been less than optimal. It is the object of this invention to provide an improved examining and scanning system which remedies these deficiencies.